Ni-NTA silica combines Ni-NTA with a macroporous silica support material optimized to suppress nonspecific hydrophobic interactions. The Ni-NTA Spin Columns provide Ni-NTA silica in a convenient microspin format for easy preparation of multiple samples in parallel. They provide a simple method for functional screening of engineered proteins, selection of clones expressing full-length translation products, and comparison of expression levels. Each spin column can purify up to 300 µg of His-tagged protein. Like all Ni-NTA matrices, Ni-NTA spin columns can be used for one-step protein purification under native or denaturing conditions.

The QIAexpressNi-NTA Protein Purification System, including the Ni-NTA Spin Columns is based on the remarkable selectivity of patented Ni-NTA (nickel-nitrilotriacetic acid) resin for proteins containing an affinity tag of six or more histidine residues — the His tag. This technology allows one-step purification of almost any His-tagged protein from any expression system under native or denaturing conditions. NTA, which has four chelation sites for nickel ions, binds nickel more tightly than metal-chelating purification systems that only have three sites available for interaction with metal ions. The extra chelation site prevents nickel-ion leaching and results in a greater binding capacity and protein preparations with higher purity than those obtained using other metal-chelating purification systems. The QIAexpress system can be used to purify His-tagged proteins from any expression system including baculovirus, mammalian cells, yeast, and bacteria.

Procedure

The purification of His-tagged proteins consists of 4 stages: cell lysis, binding, washing, and elution (see figure Ni-NTA Spin Column purification with the Ni-NTA). Purification of recombinant proteins using the QIAexpress system does not depend on the 3-dimensional structure of the protein or His tag. This allows one-step protein purification under either native or denaturing conditions, from dilute solutions and crude lysates. Up to 600 μl of a cell lysate are loaded onto a Ni-NTA spin column. A quick 2-minute spin binds the tagged protein to Ni-NTA silica, while most of the untagged proteins flow through. After a wash step, purified protein is eluted under mild conditions (such as pH reduction to 5.9, or addition of 100-500 mM imidazole) in a volume of 100-300 μl. Removal of the His tag is usually unnecessary since it is small and rarely immunogenic. The purified protein is ready for immediate use. Proteins can be purified from multiple small-scale expression cultures in approximately 30 minutes or 60 minutes (automated QIAcube procedure). Strong denaturants and detergents can be used for efficient solubilization and purification of receptors, membrane proteins, and proteins that form inclusion bodies. Reagents that allow efficient removal of nonspecifically binding contaminants can be included in wash buffers (see table). Purified proteins are eluted under mild conditions by adding 100–250 mM imidazole as competitor or by a reduction in pH.

Reagents compatible with the Ni-NTA–His interaction

Reagent

6 M guanidine HCl

8 M urea

2% Triton X-100

2% Tween 20

1% CHAPS

20 mM β-ME

10 mM DTT

50% glycerol

20% ethanol

2 M NaCl

4 M MgCl2

5 mM CaCl2

≤20 mM imidazole

20 mM TCEP

The reagents listed have been successfully used in concentrations up to those given.